Comparative genomic analysis of theIDD genes in five Rosaceae species andexpression analysis in Chinese white pear(Pyrus bretschneideri)Xueqiang Su1,*, Tiankai Meng2,*, Yu Zhao1, Guohui Li1, Xi Cheng1,Muhammad Abdullah1, Xu Sun1, Yongping Cai1 and Yi Lin1

1 School of Life Science, Anhui Agricultural University, Hefei, China2 School of Life Sciences and Technology, TongJi University, Shanghai, China* These authors contributed equally to this work.

ABSTRACTThe INDETERMINATE DOMAIN (IDD) gene family encodes hybrid transcriptionfactors with distinct zinc finger motifs and appears to be found in all higher plantgenomes. IDD genes have been identified throughout the genomes of the modelplants Arabidopsis thaliana and Oryza sativa, and the functions of many members ofthis gene family have been studied. However, few studies have investigated theIDD gene family in Rosaceae species (among these species, a genome-wideidentification of the IDD gene family has only been completed in Malus domestica).This study focuses on a comparative genomic analysis of the IDD gene family in fiveRosaceae species (Pyrus bretschneideri, Fragaria vesca, Prunus mume, Rubusoccidentalis and Prunus avium). We identified a total of 68 IDD genes: 16 genes inChinese white pear, 14 genes in F. vesca, 13 genes in Prunus mume, 14 genes inR. occidentalis and 11 genes in Prunus avium. The evolution of the IDD genes inthese five Rosaceae species was revealed by constructing a phylogenetic tree, trackinggene duplication events, and performing a sliding window analysis and a conservedmicrosynteny analysis. The expression analysis of different organs showed thatmost of the pear IDD genes are found at a very high transcription level infruits, flowers and buds. Based on our results with those obtained in previousresearch, we speculated that PbIDD2 and PbIDD8 might participate in floweringinduction in pear. A temporal expression analysis showed that the expressionpatterns of PbIDD3 and PbIDD5 were completely opposite to the accumulationpattern of fruit lignin and the stone cell content. The results of the compositephylogenetic tree and expression pattern analysis indicated that PbIDD3 and PbIDD5might be involved in the metabolism of lignin and secondary cell wall (SCW)formation. In summary, we provide basic information about the IDD genes infive Rosaceae species and thereby provide a theoretical basis for studying the functionof these IDD genes.

Subjects Agricultural Science, Bioinformatics, Genetics, GenomicsKeywords INDETERMINATE DOMAIN (IDD) genes, Chinese white pear, Phylogenetic analysis,Microsynteny, Lignin synthesis, SCW formation

How to cite this article Su X, Meng T, Zhao Y, Li G, Cheng X, Abdullah M, Sun X, Cai Y, Lin Y. 2019. Comparative genomic analysis of theIDD genes in five Rosaceae species and expression analysis in Chinese white pear (Pyrus bretschneideri). PeerJ 7:e6628DOI 10.7717/peerj.6628

Submitted 4 October 2018Accepted 15 February 2019Published 26 March 2019

Corresponding authorsYongping Cai, ypcaiah@163.comYi Lin, linyi992547404@163.com

Academic editorSheila McCormick

Additional Information andDeclarations can be found onpage 20

DOI 10.7717/peerj.6628

Copyright2019 Su et al.

Distributed underCreative Commons CC-BY 4.0

INTRODUCTIONZinc finger proteins are transcription factors with a finger-like domain that are widelydistributed in animals, microorganisms, and the plant kingdom (Miller, Mclachlan &Klug, 1985). In fact, zinc finger proteins are found in all plant eukaryotic lineages, whichindicates that these proteins might have been derived from a common eukaryoticancestor. Zinc finger proteins have been identified and functionally analysed in severalplant species, such as Jatropha curcas (Shi et al., 2018), Oryza sativa (Zhang et al., 2018),Musa acuminata (Chen et al., 2014). One group of this large family of proteins, theINDETERMINATE DOMAIN (IDD) proteins, has a highly conserved ID domain(Colasanti, Yuan & Sundaresan, 1998), which contains typical C2H2 and C2HC zinc fingermotifs (Wu et al., 2008). C2H2 zinc finger transcription factors, which are one of themost thoroughly studied transcription factor families (Agarwal et al., 2007;Wei, Pan & Li,2016), contain tandem repeat segments of approximately 30 amino acids, all of whichhave a highly conserved amino acid sequence: (F/Y)-XC-X2-5-C-X3-(F/Y)-X5-psi-X2-H-X3-5-H (wherein C and H represent cysteine and histidine, respectively, X representsany amino acid, and psi represents a hydrophobic residue) (Parraga et al., 1988).The structure obtained from this particular sequence can bind to Zn2+ and fold to form astructure with two β-sheets and an a-helix. Zn2+ mainly plays a role in the linking ofindividual amino acid chains and is also crucial to the function of zinc finger proteins(Islam, Hur & Wang, 2009; Tian et al., 2010).

The IDD protein family is a special type of C2H2 zinc finger subgroup. These proteinshave a highly conserved ID domain with an N-terminal nuclear localization signal (NLS)and four distinct zinc finger motifs (ZF1, ZF2, ZF3 and ZF4) (Colasanti et al., 2006;Kozaki, Hake & Colasanti, 2004). Two canonical zinc fingers are found in the IDD genes,and the encoded proteins also contain two unusual CCHC fingers, one of which is alsoassociated with the zinc finger domain in the Saccharomyces cerevisiae SW15 protein(Kozaki, Hake & Colasanti, 2004). Among the species for which a complete genome-wideidentification of the IDD gene family has been performed, Malus domestica has ahigher number of IDD genes than Arabidopsis thaliana, O. sativa and Zea mays(Colasanti et al., 2006; Fan et al., 2017). The functions of some IDD genes, particularlythose from A. thaliana (IDD genes with similar functions have also been reported inO. sativa and Z. mays), have been identified. The function of the IDD genes involves seedgermination (AtIDD1, 2, ZmIDD9, ZmIDDveg9) (Feurtado et al., 2011; Yi et al., 2015), leafand flower development (AtIDD4, 14, 15, 16) (Cui et al., 2013), flowering regulation(ZmID1, AtIDD8,OsID1) (Colasanti, Yuan & Sundaresan, 1998; Seo et al., 2011; Park et al.,2008), starch metabolism (AtIDD5) (Ingkasuwan et al., 2012), the regulation of plantgravitropism (AtIDD14, 15, 16, LPA1, OsIDD16, 18), root development and nitrogenmetabolism (AtIDD3, 8, 9, 10) (Jeong et al., 2015; Coelho et al., 2018).

Pear, which belongs to the Rosaceae family, is widely cultivated throughout the worldand is popular due to its unique flavour. China has a long history of eating pears, and thepears cultivated in China are mainly Pyrus bretschneideri, Pyrus pyrifolia, Pyrussinkiangensis and Pyrus ussuriensis (Vavilov, 1951; Xue et al., 2018). “Dangshan Su”

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pear (Pyrus bretschneideri cv. Dangshan Su) is a diploid pear variety that originated inDangshan County, Anhui Province, China, and due to its good quality and high medicinalefficacy (Hamauzu et al., 2007; Huang et al., 2010; Zhang et al., 2017), the fruit has avery high market value. The stone cells in pear are closely related to the flesh texture, andthe content of stone cells is a key factor in determining the quality of pear fruit (Cai et al.,2010; Yan et al., 2014). However, few studies have investigated the IDD genes duringfruit development, and the only results for Musa acuminata have been reported(Chen et al., 2014). InMusa acuminata, the expression ofMaIDD is closely related to fruitdevelopment, whereas, in Malus domestica, the IDD gene also appears to play a rolein regulating flower induction. In addition, some zinc finger proteins, such as theCCCH-tandem zinc finger protein IIP4 (Zhang et al., 2018), A. thaliana C3H14, C3H15(Chai et al., 2015) and OsIDD2 in O. sativa (Huang et al., 2018), are involved inplant secondary cell wall (SCW) formation. Although the function of several IDD geneshas been studied and their potential functions can be explained by bioinformatics,it remains unknown whether IDD genes participate in the regulation of flower induction,fruit development, lignin biosynthesis and SCW formation in Pyrus bretschneideri.

Among Rosaceae species, genome-wide identification of IDD genes has only beenperformed in Malus domestica (Fan et al., 2017). Currently, there remain many questionsabout the IDD gene family in Chinese white pear. In this study, we identified theIDD genes in five Rosaceae plants and performed a comparative genomic analysis.Woodland strawberry (Fragaria vesca) is a perennial herb with edible fruits that grows wellin most parts of the Northern Hemisphere, and its genome-wide sequencing was firstreported in 2011 (Shulaev et al., 2011). Japanese apricot (Prunus mume) has greatornamental value and in some Asian countries, the fruits of this tree are used for cookingand flavouring. Sweet cherry (Prunus avium), which originated in Europe, Anatolia,Maghreb and western Asia, is an important economic tree species (Tavaud et al., 2004),and a new report on the genome sequence of this species was published in 2017(Shirasawa et al., 2017). In addition, black raspberry (Rubus occidentalis), a specialty fruitcrop that is grown in the Pacific Northwest of the United States, has a unique flavourand rich nutritional value, and the whole-genome sequencing of this species waspublished in 2017 (VanBuren et al., 2016). These four species and Chinese white pearbelong to the Rosaceae family and might be connected through evolutionary relationships.We compared the IDD gene family of Chinese white pear with those of the fourabove-mentioned species to help us obtain a more comprehensive understanding of theIDD gene family and to further predict and analyse the functions of the IDD gene familymembers in Chinese white pear.

MATERIALS AND METHODSIdentification of IDD genes in five rosaceae plantsIn this study, the Chinese white pear genome database was obtained from the PearGenome Project (http://gigadb.org/dataset/10008). The genome databases of threeRosaceae plants (F. vesca, R. occidentalis, Prunus avium) were downloaded from genomedatabase for Rosaceae (GDR) (https://www.rosaceae.org/). The genome sequence data of

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Prunus mume were downloaded from the Prunus mume genome project (http://gigadb.org/dataset/10008). DNATOOLS software was used to create a local database containing theamino acid sequences of the IDD genes in the five Rosaceae plants (Cao et al., 2016). Toidentify the IDD genes of these five Rosaceae species, we used the following methods. First,theA. thaliana (16) andMalus domestica (20) IDD gene sequences were collected for use asquery sequences in a TBlastN search with the default E-value (Table S1). We comparedthese sequences with the local database sequences of the above-mentioned five Rosaceaespecies. Using the SMART online software program, the IDD candidate gene sequencesinitially screened by BLAST were then tested to determine whether they contained a zincfinger domain (http://smart.embl-heidelberg.de/) (Letunic, Doerks & Bork, 2012). Eachcandidate sequence containing a zinc finger domain was subsequently manually checkedfor an IDD domain, and the protein sequences lacking a full IDD domain and redundantsequences were discarded. We then isolated the candidate IDD gene sequences andused the ExPASy online site to investigate the molecular weights of the IDD proteins(http://web.expasy.org/protparam/) (Artimo et al., 2012). The subcellular localization of allIDD proteins was predicted using the WoLF PSORT website (http://www.genscript.com/wolf-psort.html) (Horton et al., 2007). Phtre2 website (http://www.sbg.bio.ic.ac.uk/phyre2)for protein three-dimensional structures prediction (Kelley et al., 2015). Gene ontology(GO) annotations analysis we use BLAST2GO software (Conesa et al., 2005).

Phylogenetic analysisSequence alignment of all IDD proteins was done using the ClustalW tool in MEGA 7.0software. The phylogenetic tree was constructed with MEGA 7.0 software using themaximum likelihood (ML) (bootstrap = 1,000) (Kumar, Stecher & Tamura, 2016).The Malus domestica IDD gene sequence used in phylogenetic tree was derived from theresults of Fan et al. (2017). The sequences of A. thaliana, O. sativa and Z. mays wereobtained from the article by Colasanti et al. (2006).

IDD gene structures and conserved motif predictionINDETERMINATE DOMAIN gene structures were compared using Gene StructureServer (http://gsds.cbi.pku.edu.cn) (Guo et al., 2007). The motifs of the IDD genes inthese five Rosaceae species were analyzed using MEME online analysis software(http://meme.sdsc.edu/meme4_3_0/intro.html) (Bailey et al., 2015). Parameters for theconserved motif prediction were motif width greater than 6 and less than 200. The numberof identified motifs was 20.

Chromosomal location, gene duplication and Ka/Ks ratio analysisFive Rosaceae species chromosome start positions and other relevant informationabout the IDD genes were obtained from the public genome database. The chromosomalphysical locations of the IDD genes of all five Rosaceae species were mapped usingMapInspect software (http://mapinspect.software.informer.com) (Hu & Liu, 2011;Lin et al., 2011; Zhu et al., 2015). To define gene duplication events, we mainly relied on thefollowing principles: (1) The similarity of the two gene-coding sequences was more

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than 80%. (2) If the two genes were located on the same chromosome and separated byat least 200 kb, we considered these two genes tandem-duplicated genes. (3) If thetwo genes were located on different chromosomes, they were called segmentallyduplicated genes (Long & Thornton, 2001). DnaSP v5.0 software was used to calculatenon-synonymous (Ka) and synonymous substitution (Ks) values and perform slidingwindow analysis. Parameters for sliding window analysis were window size 150 bpand step size nine bp (Librado & Rozas, 2009).

Microsynteny analysis and Chinese white pear IDD gene promoterCis-acting element analysisWe obtained the promoter sequence of each IDD gene from the Chinese white peargenome database, including the DNA sequence of the initiation codon (ATG) located1,500 bp upstream of each IDD gene. The online software Plantcare was used to analyzethe cis-acting elements of the promoter region (http://bioinformatics.psb.ugent.be/webtools/plantcarere/html/) (Rombauts et al., 1999). The microsynteny of six Rosaceaespecies (we added the IDD genes of Malus domestica) was identified using the MultipleCollinearity Scan toolkit (MCSscanX) (Abdullah et al., 2018).

RNA extraction and qRT-PCRThe plant material used in this experiment was the “Dangshan Su” pear, which grows inthe Yuanyichang agricultural park (Dangshan County, Anhui Province, China). Samplesof current-year flowers, flower buds, stems, mature leaves and fruits were obtainedfrom 40-year-old Pyrus bretschneideri cv. Dangshan Su trees. The fruits were picked 15days after pollination (DAP), 39 DAP, 47 DAP, 55 DAP, 63 DAP, 79 DAP and 145 DAP,and the fruits obtained 39 DAP were used for the expression analysis in differentorgans. “Dangshan Su” pear buds were treated with gibberellin (GA) and sucrose.Specifically, on a clear morning, terminal buds on current-year spurs were sprayed (<5 cm)with GA (700 mg·L-1) and sucrose (20,000 mg·L-1) using a low-pressure hand wandsprayer. Some plant materials were collected immediately after spraying with GA andsucrose and stored at -80 �C for use as controls. Materials collected 2 h (H), 4 H, 6 H, 8 H,12 H and 24 H After GA and sucrose treatment were stored at -80 �C for gene expressionanalyses and samples collected 0 h post-treatment (HPT), 2 HPT, 4 HPT, 6 HPT,8 HPT, 12 HPT and 24 HPTwere also collected. RNAwas extracted from the plant materials(including fruits and various organs) using a plant RNA extraction kit from Tiangen(Beijing, China). Reverse transcription was performed using a PrimeScriptTM RT reagent kitwith gDNA Eraser (Takara, Kusatsu, Japan), and each reaction consisted of one mg of RNA.The qRT-PCR primers for the pear IDD genes were designed using Beacon Designer 7software (Table S8). Each 20-mL qRT-PCR system consisted of 10 mL of SYBR� Premix ExTaqTM II (2�) (Takara, Kusatsu, Japan), two mL of cDNA, 6.4 mL of water and 0.8 mL ofPbIDD-F and PbIDD-R. The pear Tubulin gene (No. AB239680.1) (Wu et al., 2012)was used as an internal reference. The reaction procedure was performed following theinstruction manual, and three repetitions were run for each sample. The relativeexpression levels were calculated using the 2-DDCt method (Livak & Schmittgen, 2001).

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RESULTSIdentification, characterization and phylogenetic analysis of IDD genesA total of 68 IDD proteins were identified and used for further analysis (Table 1; Table S1),which included 16 IDD proteins (PbIDD1-PbIDD16) in Chinese white pear. We alsoidentified a total of 52 IDD proteins in the other four species, including 14 in F. vesca(FvIDD1-FvIDD14), 13 in Prunus mume (PmIDD1-PmIDD13), 14 in R. occidentalis(RoIDD1-RoIDD14) and 11 in Prunus avium (PaIDD1-PaIDD11). Although only 11 IDDproteins were identified in Prunus avium, the numbers of IDD proteins in the other specieswere very similar but fewer than the number of IDD proteins in Malus domestica (20).We used ExPASy software to calculate the physicochemical parameters of the IDD genes(Table S2). We identified 68 IDD genes with isoelectric points (pIs) greater than 7. In theseRosaceae species, the lowest pI value was 7.76 (PaIDD8), whereas the highest pI valuewas 9.45 (FvIDD4). The molecular weights of these IDD genes were quite different, rangingfrom 42.0 kDa for PaIDD10 to 79.6 kDa for PmIDD8. The numbers of amino acids in theIDD proteins were also quite different. PbIDD14 contained the fewest (only 381)amino acids, whereas, PmIDD8 had the largest number (728). The grand averagehydropathicity (GRAVY) of all 68 IDD genes was less than 1, and the smallest GRAVYvalue (-0.914) was obtained for PbIDD15.

A phylogenetic tree of 98 IDD proteins was constructed using the ML method(Fig. 1). Based on the phylogenetic tree, we identified four phylogenetic groups(groups 1–4). Group 1 had the most members and contained all six Rosaceae species and

Table 1 Basic Information of IDD Gene in Pyrus bretschneideri.

Gene name Gene ID AA KD pI GRAVY Preditcedsubcellularlocalization

PbIDD1 Pbr029706.1 489 51.5 8.17 -0.460 nucl

PbIDD2 Pbr006167.1 535 55.7 8.98 -0.427 nucl

PbIDD3 Pbr032492.1 533 58.0 8.98 -0.659 nucl

PbIDD4 Pbr021137.1 553 60.4 9.31 -0.634 nucl

PbIDD5 Pbr028264.1 526 57.4 8.89 -0.693 nucl

PbIDD6 Pbr019853.1 537 59.1 8.67 -0.807 nucl

PbIDD7 Pbr012193.2 567 61.7 8.92 -0.772 nucl

PbIDD8 Pbr020403.1 524 57.3 9.07 -0.740 nucl

PbIDD9 Pbr009954.1 465 50.9 9.19 -0.664 nucl

PbIDD10 Pbr012907.1 465 51.1 8.99 -0.629 nucl

PbIDD11 Pbr008330.1 464 51.0 9.35 -0.727 nucl

PbIDD12 Pbr012192.1 607 64.7 9.13 -0.723 nucl

PbIDD13 Pbr025606.1 426 47.2 9.25 -0.646 nucl

PbIDD14 Pbr029012.1 381 42.7 9.36 -0.655 nucl

PbIDD15 Pbr038802.1 502 56.4 9.02 -0.914 nucl

PbIDD16 Pbr012170.1 492 54.4 8.93 -0.805 nucl

Note:The IDD genes of Pyrus bretschneideri identified in this study are listed.

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44 IDD genes: Pyrus bretschneideri (8), F. vesca (7), Prunus mume (6), R. occidentalis (7),Prunus avium (6), Malus domestica (10). Groups 3, 2 included 21 and 14 IDD genes,respectively. Group 4 had the fewest members: two IDD genes in Pyrus bretschneideri,two IDD genes in F. vesca, two IDD genes in Prunus mume, two IDD genes inR. occidentalis, one IDD gene found in Prunus avium and no IDD gene in Malusdomestica. ZmID1 was grouped into a class by itself, and most PbIDD genes were moretightly grouped with MdIDDs. In addition, the IDD genes of some Rosaceae species wereclosely related to those of A. thaliana and O. sativa: PbIDD3, PbIDD5, FvIDD3,RoIDD4, PmIDD3 and PaIDD4 were included in group 1 and were closely related toOsIDD2; FvIDD12, RoIDD9, PmIDD11, PaIDD5 and AtIDD3 and 8 are closely related, andAtIDD1 was included in group 3 and clustered with seven Rosaceae IDD genes. The resultsof GO annotations (Table S9) showed that all 68 IDD genes had the function of nucleicacid binding (GO:0003676).

Structural and conserved motif analysis of IDD proteinsTo more comprehensively analyse the structural diversity of the IDD genes in the fiveRosaceae species, we created exon-intron pattern maps of all 68 IDD genes (Fig. 2). A totalof 25 of the 34 IDD genes in group 1 had three exons and the other six genes hadtwo exons. Additionally, FvIDD7 (4), RoIDD6 (5) and PmIDD8 (6) had a large number ofexons. The analysis of group 2 revealed that FvIDD11 had two exons, PbIDD7 hadfour exons and all the other members had three exons. Group 3 comprised a subfamilywith a complex gene structure: 11 genes had four exons and four genes had three exons.

Figure 1 Phylogenetic relationships and subfamily designations in IDD proteins from Pyrusbretschneideri, Fragaria vesca, Prunus mume, Rubus occidentalis, Prunus avium, Malus domestica,Arabidopsis thaliana, Oryza sativa. Phylogenetic relationships and subfamily designations in IDDproteins from Pyrus bretschneideri, Fragaria vesca, Prunus mume, Rubus occidentalis, Prunus avium,Malus domestica, Arabidopsis thaliana,Oryza sativa and Zea mays. Groups 1–4 are represented by shadesof red, green, blue and cyan, respectively. Full-size DOI: 10.7717/peerj.6628/fig-1

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All members of group 4 had a gene structure comprising three exons. The lack ofhigh similarity among the 68 IDD genes allowed a better understanding of the conservedmotifs in these IDD genes. We used MEME software to identify 20 conserved motifs(Fig. 2; Table S3). Motifs 2, 3, 1 and 4 encoded the ID domain, whereas the remainingmotifs did not have functional annotations. As shown in Fig. 3, motifs 2, 3, 1, 4 representedthe zinc fingers ZF1, ZF2, ZF3 and ZF4, respectively. The four conserved motifsconstituted a conserved ID domain, whereas these four conserved motifs represented twoC2H2 and two C2HC structures (ZF1 and ZF2 belonged to C2H2, and ZF3 and ZF4belonged to C2HC). Two C2H2 and two C2HC structures were identified through asequence alignment of the conserved ID domains of five species and the results showedthat the ID domain was highly conserved. The cysteine (C) and histidine (H) residues ineach zinc finger domain were conserved in each species, consistent with the resultsof previous studies (Figs. S1–S5). All 68 IDD genes had motifs 1–4, which were the mostconserved. In addition, some members of the same group and the more closely relatedmembers had highly similar motif compositions (e.g., FvIDD7 and RoIDD6 in group 1;PmIDD10 and PaIDD9 in group 2). These genes were closely related and had exactly the

Figure 2 Predicted Pyrus bretschneideri, Fragaria vesca, Prunus mume, Rubus occidentalis and Prunus avium IDD protein conserved motifsand exon-intron structures. (A) Gene structures of the IDD genes. Black wedge indicates exon, black line indicates intron and red wedge indicatesUTR. (B) Distribution of MEME motifs in IDD genes. (C) The color and corresponding number of each motif box.

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same motif compositions. We also identified certain group-specific motifs, such as motifs12, 17, 20 in members of group 4 and motif 19 in some members of group 1.

We also identified a NLS in the N-terminal border of these IDD genes (Fig. S6).According to previous studies, this NLS is usually KKKR or KRKR (Colasanti et al., 2006).Not all members in the five Rosaceae species, PbIDD13, 14, FvIDD10, 11, 12 andPmIDD11, have a NLS. In addition to the conserved IDD domain, we also found twoC-terminal domains (MSATALLQKAA and TRDFLG), which are encoded by motifs 6, 7,respectively, in some members (Fig. 2; Figs. S1–S5). All nine members of group 4 lackedboth motifs 6 and 7. Among the members of group 1, PaIDD10 did not containmotif 6, and PbIDD14 and PmIDD8 lacked motif 7. All members of groups 2, 3 containedthese two motifs. The lack of these two conserved sequences at the C terminus ofsome IDD genes might explain why the members of group 4 were clustered into a singlecategory and might also underlie the differences among the functions of these proteins(Fig. 1).

Chromosomal location and duplication events of IDD family genes inrosaceaeBased on the complete genome sequences of the five Rosaceae species, the exactchromosomal locations of all 68 IDD genes were identified (Fig. 4). In Pyrus bretschneideri,the 16 IDD genes were distributed on chromosomes 3, 4, 8, 9, 11, 12, 14, 15, 16, 17.In F. vesca, the IDD genes were distributed on chromosomes 1, 2, 3, 4, 5, 6. In

Figure 3 Conserved ID domain composition. All 68 IDD genes had a characteristic ID domain. The ID domain consists of four zinc fingers(Z1, Z2, Z3 and Z4). Alignment analysis of the 68 IDD gene sequences using the ClustalW tool in MEGA 7.0 software. These domain diagrams wereplotted using the online WebLogo tool. Full-size DOI: 10.7717/peerj.6628/fig-3

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R. occidentalis, the IDD genes were mainly distributed on chromosomes 2, 4 and 6. Severalother IDD genes were distributed on chromosomes 1, 3 and 5. In Prunus mume, exceptPaIDD5, which could not be mapped to any chromosome, the IDD genes weredistributed on chromosomes 1, 2, 4, 6, 7, 8. However, in Prunus avium, four IDD geneswere distributed on chromosome 1 and the remaining 7 IDD genes were distributedon chromosomes 3, 5, 6, 7, 8. Only four pairs of duplicated genes were identifiedin pear (Fig. 4). All duplicated genes identified in Pyrus bretschneideri were segmentalduplications.

To clarify the driving forces of gene duplication and explore the impact of these geneson evolutionary processes, we calculated the Ka, Ks and Ka/Ks ratio for the four duplicatedgene pairs in Pyrus bretschneideri. Ka/Ks = 1 is the cut-off value that indicatesneutral selection, Ka/Ks < 1 represents negative selection and Ka/Ks > 1 represents positiveselection (Bitocchi et al., 2017). For all four duplicated gene pairs identified in Pyrusbretschneideri, the Ka/Ks values were less than 0.2726 (Table S4). In the evolutionaryprocesses of genes, positive selection may be overshadowed by strong negativeselection (Han et al., 2016). Therefore, to comprehensively explain the selection pressure ofIDD genes, we performed a sliding window analysis of the four pairs of IDD paraloguesin pears (Fig. S7). Most coding site Ka/Ks ratios were less than one, with exceptions

Figure 4 Chromosomal locations of five Rosaceae species. Chromosomal locations of IDD genes in Pyrus bretschneideri (A), Fragaria vesca (B),Rubus occidentalis (C), Prunus mume (D) and Prunus avium (E). Duplicated gene pairs are connected with colored lines.

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for one or several distinct peaks (Ka/Ks > 1). The Ka/Ks ratios of the conserved IDDdomains were less than 1.

Microsynteny and evolutionary analysis of the IDD gene familyFor the identification of interspecific microsynteny, we visualized the location ofhomologous and orthologous genes. A total of 30 orthologous gene pairs were identified infive Rosaceae species (Fig. 5; Table S5) and these included one, eight, three, six and12 collinear gene pairs between Pyrus bretschneideri and F. vesca, Prunus mume,R. occidentalis, Prunus avium, Malus domestica, respectively. Notably, PbIDD2 alsoshowed a collinear relationship with the members of the MdIDD, PmIDD and PaIDDfamilies. In contrast, two IDD genes from pear matched one pair of Prunus mume or

Figure 5 Microsynteny of regions among Pyrus bretschneideri, Fragaria vesca, Prunus mume, Rubusoccidentalis, Prunus avium and Malus domestica. The chromosome numbers are indicated by differ-ently colored boxes and are labelled by Pb, Fv, Pm, Ro, Pa and Md. The differently colored boxes alsorepresent the sequence lengths of chromosomes in megabases. The black line indicates the syntenicrelationship among the IDD regions. Full-size DOI: 10.7717/peerj.6628/fig-5

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R. occidentalis genes. For example, PbIDD9 and PbIDD10 were orthologous to PmIDD9,whereas PbIDD9 and PbIDD10 were orthologous to RoIDD7.

However, we did not identify any orthologous gene pairs between Pyrus bretschneideriand O. sativa, Z. mays and A. thaliana. We also compared the other four Rosaceae specieswith these three species, and the results did not reveal any orthologous gene pairs.To further study the evolution of IDD genes, we collected the IDD genes from fourmonocotyledons (M. acuminata, Sorghum bicolor, Z. mays and O. sativa) and twodicotyledons (Vitis vinifera and A. thaliana). A phylogenetic tree of the genomes of these12 species was obtained to perform an evolutionary analysis of the IDD gene family(Fig. S8A). Several Rosaceae species exhibit a closer relationship and are more distant frommonocots. This result is basically consistent with the results of the phylogenetic treeconstructed from all IDD genes of 12 species (Fig. S8B). The results identified 12 classes(classes I–XII). The Pyrus bretschneideri and Malus domestica, Prunus avium and Prunusmume IDD genes often cluster into one class, and the IDD genes of monocotyledonsare closely related (i.e., classes X and XI are only composed of IDD genes ofmonocotyledons). ZmID1, OsID1 and SbID1 are closely related, but we did not identifyan orthologous gene for the ID1 gene in several other species. Class III contained all speciesexcept Malus domestica, and the IDD genes in this subfamily did not have completeMSATALLQKAA and TRDFLG domains.

Analysis of Cis-acting elements in the promoters of the IDD genes inChinese white pearPlant IDD genes may be involved in multiple biological processes, and hormones affecttheir expression. We predicted the cis-acting elements of 16 Chinese white pear IDDgene promoters (Fig. 6; Table S7). A total of 13 IDD genes (except PbIDD7, 9, 10)contained at least one G-Box, which is a light-responsive element, indicating that the

Figure 6 Promoter Cis-elements of the 16 PbIDDs. Potential Cis-elements in the 5′ regulatorysequences of the 16 PbIDDs. Full-size DOI: 10.7717/peerj.6628/fig-6

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expression of these genes may be regulated by light. Eight Pyrus bretschneideri IDD geneshad HSE, and 11 members contained MBS components. TC-rich repeats, which arecis-acting elements involved in defence and stress responsiveness, were identified inPbIDD1, 6, 7, 9, 10, 12, 14 and 16. In addition, there were many cis-acting elements relatedto the responses to hormones, including responses to Abscisic acid response element(ABRE), methyl jasmonate (MeJA) (CGTCA-motif, TGACG-motif), GA (P-box,GARE-motif), auxin (TGA-element), and salicylic acid (TCA-element). The auxin-relatedTGA-element was identified in only five members (PbIDD1, 2, 4, 8, 13), which was the leastnumber of elements components found. A total of 41 Cis-acting elements associated withMeJA were found in nine members, and these were the most abundant Cis-actingelements. Cis-acting elements responding to GA were the most widespread, and twelve ofthe Pyrus bretschneideri 16 IDD genes had this element. The related Cis-acting elements ofabscisic acid and salicylic acid were identified in 10 and nine members, respectively. Wefound that PbIDD1 has Cis-acting elements related to the above five hormones, whereasPbIDD9 only has cis-acting elements that are responsive to MeJA. Of the 16 pear IDDgenes, 12 contained 19 CGTCA-motifs and TCA-elements appeared in the promoterregions of 10 members, for a total of 22 genes. In addition, the CAT-box and theCCGTCC-box were found in 11 members (PbIDD1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 14); these areCis-acting elements related to meristem expression.

Expression characteristics of Chinese white pear IDD genesTo obtain a more in-depth understanding of the Chinese white pear IDD gene family,we investigated the expression patterns of PbIDDs in different organs (Fig. 7). No PbIDD14expression was detected in any of the organs of Chinese white pear. Among the remaining15 members, PbIDD4, 9, 11 were highly expressed in buds but showed extremelylow expression in other organs. PbIDD1 and 10 were mainly expressed in flowers. PbIDD2,6, 8 and 15 were mainly expressed in fruits, flowers and buds. Among these four genes,PbIDD2 and 8 showed the highest expression levels in buds, whereas PbIDD6 and15 exhibited the highest expression levels in fruit. PbIDD5, 12, and 16 were detected inmultiple organs, but the main difference in the expression of these genes was found inthe leaves: (1) PbIDD12was highly expressed in leaves; (2) although PbIDD5was expressedin all five organs, its lowest expression was detected in the leaves; and (3) PbIDD16showed almost no expression in leaves. PbIDD3, 7 and 13 were mainly expressed in thefruit of Chinese white pear, and PbIDD3 and 7 showed almost no expression inother organs.

We investigated the expression patterns of 16 IDD genes in Chinese white pear fruit atseven developmental stages (Fig. 7). The expression level of PbIDD1 peaked at 39 DAPand was low during the other six fruit developmental stages. The expression peaks ofPbIDD2, 7, 8, 11, 13, 16 also appeared at 39 DAP, but high expression of these genes wasalso detected at the early developmental stages (15 and 47 DAP). At the late developmentalstages of fruit (79 and 145 DAP), these genes were expressed at a moderate level.PbIDD6, 15 exhibited a similar expression pattern as the above-mentioned genes, with theexception that PbIDD6, 15 were detected at the early developmental stages of fruit

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Figure 7 Expression patterns of IDD genes of Chinese white pear in different organs and in fruit atdifferent developmental stages. Expression patterns of IDD genes in Chinese white pear in differentorgans (A–O). Expression patterns of IDD genes in Chinese white pear at different developmental stages(P–DD). �significant difference at P < 0.05, ��significant difference at P < 0.01.

Full-size DOI: 10.7717/peerj.6628/fig-7

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but were expressed at extremely low levels at the late developmental stages of fruit.The expression level of PbIDD4 was higher only at the early stage of fruit development(15–55 DAP) and at 79 DAP. PbIDD9 had a high transcription level at 63 and 145 DAP,and PbIDD12 was mainly expressed at 79 DAP. PbIDD10 had two expression peaks, one at39 DAP and another at 55 DAP. PbIDD3 and 5 had the same expression pattern:these two genes were mainly expressed at the early stage of fruit development, and theirexpression level then gradually decreased.

Gibberellin and sugar response pattern analysis of PbIDDsChinese white pear PbIDD2, 5, 6, 8, 9, 12, 16 are mainly expressed in bud and flower.We treated the bud of Chinese white pear with exogenous GA and sucrose to investigatethe expression pattern of PbIDD2, 5, 6, 8, 9, 12, 16 under GA and sucrose treatment(Fig. S9). Under exogenous GA treatment, PbIDD2, 5, 6, 8, 9, 12, 16 showed three differentexpression patterns (activation, inhibition and invariance). There was no significantchange in the transcriptional level of PbIDD5, 9 under exogenous GA treatment.The expression levels of PbIDD2, 8 continued to decrease after exogenous GA treatment,indicating that the expression of PbIDD2, 8 was inhibited by GA. PbIDD6, 12 showed asignificant increase in expression after GA treatment (reaching a peak at 3HPT);thereafter, the expression level gradually decreased to untreated levels. PbIDD16 reachedthe maximal expression levels at 2, 4HPT. Overall, the expression of PbIDD6, 12, 16showed activation by GA. The transcription levels of PbIDD2, 8 increased gradually aftersucrose treatment in Chinese white pear bud. The expressions of PbIDD5, 12, 16 were notalways induced by sucrose. The lowest expression levels of PbIDD5, 12 were foundat 4HTP, while the lowest expression level of PbIDD16 was observed at 1HTP.The expressions of PbIDD6, 9 were inhibited by sucrose.

DISCUSSIONIDD gene family encode hybrid transcription factors that have four zinc finger structuresand one nuclear positioning signal. IDD proteins play important roles in regulating plantflowering, development, stress resistance, secondary metabolism and other processes(Wong & Colasanti, 2007; Matsubara et al., 2008). The plant IDD gene can also regulatethe expression of the key enzyme genes of lignin synthesis, which affects the biosynthesis oflignin and regulates the biosynthesis of SCW formation (Huang et al., 2018).

In this study, we identified 68 IDD genes in five Rosaceae species (Table 1; Table S2).The pI values of all IDD protein were greater than 7, indicating that all these proteinsare alkaline proteins. The proteins were all hydrophilic, as demonstrated by the findingthat their GRAVY values were less than 1. Among the Rosaceae species, Pyrusbretschneideri had the second greatest number of IDD gene family members after Malusdomestica (Fan et al., 2017), and this difference might be traced back to the origin ofthe IDD gene family, during which time the numbers of IDD genes in Pyrus bretschneideriandMalus domesticawere different. Previous studies have shown that plant whole-genomeduplication (WGD) and chromosome rearrangement alter the sequences of genes, and thisalteration is accompanied by widespread gene loss (Adams & Wendel, 2005; Tang et al.,

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2008). The common ancestor of Rosaceae species has nine chromosomes (Shulaev et al.,2011; Wu et al., 2013). Pyrus bretschneideri and Malus domestica both experienced twoWGDs 130 million years ago (Mya) and 30–45 Mya, but the number of chromosomes wasonly 17 (Jaillon et al., 2007; Guo et al., 2013;Wu et al., 2013). This finding indicates that thenine chromosomes of Rosaceae species ancestors experienced doubling, breaking andfusion during the long process and ultimately formed the 17 chromosomes of Pyrusbretschneideri and Malus domestica. During this process, the genome of a species mightbecome unstable and prone to gene replacement, leading to chromosome rearrangementand gene loss (Paterson, Bowers & Chapman, 2004). It is possible that the IDD genesin Pyrus bretschneideri were lost during this process, which explains why the number ofIDD genes in Pyrus bretschneideri was less than that inMalus domestica. The phylogenetictree divided 68 IDD genes into four groups (Fig. 1). ZmID1 comprised a separateclass in the phylogenetic tree, and we did not identify a closely related gene (Fig. 1).At present, ZmID1 homologous genes have been identified only in O. sativa and S. bicolor,which belong to the same family of Gramineae (Colasanti et al., 2006). This finding mightindicate that the ID1 gene is unique to Gramineae plants. Interestingly, five Rosaceaespecies had at least one IDD gene present in each clade, and this finding had strongbootstrap support. These results showed that the rapid duplication of IDD genes occurredbefore the divergence of these Rosaceae species.

The gene structure and conserved motif composition might be closely related tothe diversity of gene functions (Swarbreck et al., 2008). Genes in the same subfamily tendto have very similar gene structures, which indicates that these genes might havesimilar functions (Fig. 2). For example, PbIDD12 and PmIDD10 in group 2 had the samegene structure (three exons and two introns), and their exon lengths were almost the same.Furthermore, based on the MEME analysis (Fig. 2), we found that members of thesame subfamily had roughly the same conserved protein motifs, but some differencesin motif composition were found between different subfamilies. An N-terminal NLS isabsent in many Rosaceae IDD genes (Fig. 6). All IDD genes in A. thaliana have aNLS. OsIDD10, which belongs to the O. sativa IDD gene family, also lacks a NLS(Colasanti et al., 2006). The results indicate that the lack of NLS sequence in somemembers of the IDD gene family might be due not to the mutation or deletion of this entirecharacteristic sequence but rather to an extensive deletion of the N-terminal sequence.The ancestors of the special IDD genes in these Rosaceae species might have appeared afterthe differentiation of these Rosaceae species from the common ancestors of A. thaliana,and some of the N-terminal sequences, which includes the NLS, were lost after theevolutionary process. In addition to the highly conserved ID domain, we found two smalldomains (Figs. S1–S5) in the C-terminal region (except in members of group 4).The MSATALLQKAA and TRDFLG motifs are conserved regions outside the ID domainbut are not characteristically conserved domains of IDD genes. Obviously, the membersof group 4 lost these two small domains during evolution, which resulted in thegeneration of functional differences. We found that group 4 contained genes in A. thaliana(AtIDD15) and O. sativa (OsIDD14). AtIDD14, 15, 16 synergistically regulate spatial

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auxin biosynthesis by direct targeting YUCCA5 (YUC5), TRYPTOPHANAMINOTRANSFERASE of ARABIDOPSIS1 (TAA1) and PINFORMED1 (PIN1) andthereby controlling aerial organ morphogenesis and gravitropic responses in plants(Cui et al., 2013). OsIDD14 is the functional orthologue gene of AtIDD15 and plays arole in the regulation of shoot gravitropism in O. sativa. Members of this groupmight have evolved to have functions in regulating the geotropism of plant organs.However, this group does not contain Malus domestica IDD genes, which indicates thatMSATALLQKAA and TRDFLG domains might have been preserved during theevolutionary process or that IDD genes do not play a role in the regulation of organgeotropism in Malus domestica.

The functional divergence of genes often occurs after gene duplication and isaccompanied by the introduction of new functions or the loss of original functions(Chao et al., 2017). In addition, gene duplication events are the main driving force for genefamily expansion and thus constitute an important method used by plants to adapt tochanging climates and environments (Tang et al., 2016). Gene duplication events havebeen identified in multiple gene families, such as the PKS gene family in Gossypiumhirsutum (Su et al., 2017), the HSF gene family in Sesamum indicum (Dossa, Diouf & Cisse,2016), and the WRKY gene family in Musa acuminate (Goel et al., 2016). A total offour PbIDD gene pairs were identified as duplications in this study, and these duplicatedgene pairs were all created by segmental duplication (Table S4). Four pairs of segmentallyduplicated genes were also identified in Malus domestica, verifying the reliability ofour results. However, we did not identify gene duplication events in F. vesca, Prunusmume, R. occidentalis or Prunus avium. This finding might be due to the fact that F. vesca,Prunus mume, R. occidentalis and Prunus avium underwent only one WGD, but Pyrusbretschneideri had a recent WGD in addition to an old WGD (Zhang et al., 2012;Wu et al., 2013). The four pairs of duplicated genes that appeared in pears exerted a verypositive effect on the expansion of the IDD gene family and contributed to thefunctional diversification of IDD genes. We then performed a sliding window analysisof the four pairs of segmentally duplicated genes in Chinese white pear, and the resultsshowed that these genes experienced intense purifying selection to maintain theirfunctional stability.

We did not identify the orthologous gene pair of IDD genes between severaldicotyledons and monocotyledons. Therefore, we preliminarily hypothesized thatIDD genes might have appeared after the differentiation of the common ancestors of thesespecies differentiated. After the differentiation of monocotyledons and dicotyledons,two canonical zinc fingers and two unusual CCHC fingers formed a special zinc fingerdomain (ID domain), which defines IDD genes in all plants. Based on the results, we drewa hypothetical IDD genes evolutionary model map of Rosaceae species (Fig. S10).The ancestors of dicotyledons evolved through complex evolutionary processes to produceRosaceae species. After an old WGDs, F. vesca, R. occidentalis, Prunus avium and Prunusmume formed the current IDD gene family. Malus domestica and Pyrus bretschneideriformed the IDD gene family after two WGDs. Subsequently, Pyrus bretschneideriand another Rosaceae species differentiated into two IDD gene types (with or without the

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MSATALLQKAA and TRDFLG domains). The IDD gene lacking these two domainsmay have derived the function of regulation of plant organ geotropism. Malus domesticadid not detect such genes, perhaps becauseMalus domestica does not rely on IDD genes toparticipate in the regulation of organ geotropism.

Previous research has shown that IDD genes are most highly expressed in leaves andbuds (Fan et al., 2017), but the highest pear IDD gene expression was detected in fruit,flower and bud organs. PbIDD7 and MdIDD19 represent a pair of homologous genesthat are closely related.MdIDD19 was mainly expressed in the leaf, fruit and bud, whereasPbIDD7 was mainly expressed in fruit and bud organs and showed almost no expression inleaves (Fig. 7). The difference between the two homologous gene expression patternswas mainly concentrated in the leaf expression pattern. Many IDD genes are associatedwith leaf development and photosynthesis. However, we did not identify cis-actingelements related to the light response in the promoter region of PbIDD7 (Fig. 6), whereasabundant light-responding elements have been found in the promoter region ofMdIDD19(Fan et al., 2017). This finding might be the main reason for the large difference inthe expression patterns between PbIDD7 and MdIDD19 in leaves. We found that PbIDD4showed a high transcription level only in buds but exhibited a very low expressionlevel in fruits and other organs (Fig. 7). This gene might not be the main gene involved invarious physiological metabolism in fruit but plays an important role in the developmentof buds. Interestingly, we found that PbIDD2, 5, 6, 8, 9, 12, 16 showed highertranscription levels in flowers and buds, although the expression in buds was higher thanthat in flowers. In A. thaliana, O. sativa, Z. mays, IDD genes, such as AtIDD8, ZmID1,and OsID1, are involved in plant flower induction and are regulated by hormonesand sugars (Matsubara et al., 2008; Wu et al., 2008; Jeong et al., 2015). GA can regulateplant flowering by inhibiting flowering genes (Zhang et al., 2016; Galvao et al., 2012).The promoter regions of PbIDD2, 6, 8, 12, 16 contain the GA-response cis-acting element.Chinese white pear buds were treated with exogenous GA, and the resulting expressionpatterns of PbIDD2, 5, 6, 8, 9, 12, 16 were detected, which revealed that these geneswere mainly expressed in the buds and flowers of Chinese white pears (Fig. S9). PbIDD5, 9were not induced by GA, probably because these genes do not contain GA-relatedcis-acting elements. After exogenous GA treatment, the expression level of PbIDD2, 8decreased continuously, indicating that these genes were inhibited by GA. However, theexpression levels of these two genes increased gradually after sucrose treatment,indicating that PbIDD2, 8 were induced by sucrose. We also identified a large number oflight-responsive cis-acting elements in the promoter regions of PbIDD2, 8, whichsuggested that these two genes might respond to photoperiodic signals. Similar results havebeen found in Malus domestica: MdIDD7 is inhibited by GA in response to sucrose(Fan et al., 2017). In summary, the results suggested that PbIDD2 and 8 might participatein the GA flowering pathway during physiological flower bud differentiation and regulatethe plant flowering process in response to sugar and photoperiod signals. PbIDD1and 10 were mainly expressed in the flowers of Chinese white pear, probably because thesetwo genes are involved in the synthesis of bioactive substances in flowers.

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The stone cells and lignin in “Dangshan Su” pear fruit are mainly formed duringthe period from 15 to 63 DAP and reach their peak levels at 55 DAP (Zhang et al., 2017).The expression analysis of these 16 PbIDDs at seven developmental stages of fruit showedthat the expression pattern of any one gene was not consistent with the trends obtained forthe “Dangshan Su” pear fruit stone cell and lignin contents (Fig. 7). However, there weretwo additional specific genes (PbIDD3 and PbIDD5) and the expression patterns ofthese two genes showed completely opposite trends compared with the accumulation of“Dangshan Su” pear stone cells and lignin. PbIDD3 and 5 showed extremely highexpression levels at 15 DAP. From 15 to 47 DAP, which is a stage characterized by themassive accumulation of lignin and stone cells, the expression of these two genes graduallydecreased. At the peak of lignin accumulation (47, 55 and 63 DAP), PbIDD3 and 5showed very low transcription levels, whereas at the late stage of fruit development(79 and 145 DAP), the expression levels of these genes increased slightly. According to thephylogenetic tree (Fig. 1), PbIDD3, PbIDD5 and OsIDD2 were the most closely relatedgenes. In O. sativa, OsIDD2 regulates SCW formation while negatively regulatingthe expression of genes encoding key lignin synthesis enzymes, such as cinnamyl alcoholdehydrogenase (CAD), to inhibit lignin biosynthesis (Huang et al., 2018). In Chinese whitepear, CAD is predicted to regulate the biosynthesis of fruit lignin. The expressionpattern of PbCAD is exactly the same as the accumulation patterns of stone cells and lignin(Cheng et al., 2017). The expression patterns of PbIDD3 and PbIDD5 were opposite tothose found for PbCAD. Moreover, PbIDD3 and PbIDD5 expression was low during thehigh expression period of PbCAD and was higher during the low expression period ofPbCAD. In addition, PbIDD3 and PbIDD5 exhibited extremely high transcription levels infruit. Subsequently, we predicted the three-dimensional structures of PbIDD3, 5 andOsIDD2 (Fig. S11). As shown in Fig. S11, the three-dimensional structures of PbIDD3, 5exhibit high similarity with that of OsIDD2, indicating that these proteins might havesimilar functions and molecular mechanisms in regulating downstream gene expression.We speculate that PbIDD3, 5 are mainly expressed in fruit and have similar functionsto OsIDD2 in regulating SCW formation in pear fruit cells and inhibiting ligninbiosynthesis by inhibiting the expression of the genes encoding key lignin synthesisenzymes. In future studies, the overexpression and gene silencing of PbIDD3, 5 in“Dangshan Su” pear will be performed to determine their functions, and the interactionsbetween PbIDD3, 5 and PbCAD will be explored through a yeast one-hybrid assay.

CONCLUSIONWe identified 68 IDD genes in five Rosaceae species (Pyrus bretschneideri, F. vesca,Prunus mume, R. occidentalis and Prunus avium), which we systematically assessed bybioinformatic analysis. According to the phylogenetic tree, the 68 IDD genes were dividedinto four groups. In each class, we found that the structures of the genes and thecompositions of the conserved motifs were very similar. Through a series of bioinformaticsanalyses, we explained the possible evolutionary patterns of IDD genes in these Rosaceaespecies. According to qRT-PCR, Chinese white pear IDD genes have high expressionlevels in fruit, flower and bud. Further, PbIDD2, 8 are mainly expressed in the buds of

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Chinese white pear and are responsive to the induction of GA and sucrose. That PbIDD3, 5are may be involved in the regulation of SCW formation and lignin biosynthesis in Chinesewhite pear fruit. All in all, this study reveal the basic information of the five Rosaceaespecies’ IDD genes and predicts the potential functions of some pear IDD proteins.These results will provide an important theoretical basis for improving the quality of“Dangshan Su” pear.

ACKNOWLEDGEMENTSThe authors are deeply grateful to Prof. Yongping Cai and Prof. Yi Lin, who provided thesample used in the study and very effective direction. The authors also thank Dr. Xu Sun,Dr. Muhammad Abdullah, Dr. Xi Cheng, Dr. Guohui Li, Dr. Yu Zhao for providingvaluable suggestions and comments. Finally, author would like to thank Dr. Meng forhis help in revising the paper.

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis study was supported by the National Natural Science Foundation of China(Grant #31640068), the Anhui Provincial Natural Science Foundation (Grant#1808085QC79) and the Graduate innovation fund of Anhui Agricultural University(Grant #2018yjs-41). The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Grant DisclosuresThe following grant information was disclosed by the authors:National Natural Science Foundation of China: #31640068.Anhui Provincial Natural Science Foundation: #1808085QC79.Graduate innovation fund of Anhui Agricultural University: #2018yjs-41.

Competing InterestsThe authors declare that they have no competing interests.

Author Contributions� Xueqiang Su conceived and designed the experiments, performed the experiments,contributed reagents/materials/analysis tools, prepared figures and/or tables, authoredor reviewed drafts of the paper, approved the final draft.

� Tiankai Meng analyzed the data, authored or reviewed drafts of the paper, modifyour language problem.

� Yu Zhao conceived and designed the experiments, performed the experiments,contributed reagents/materials/analysis tools, prepared figures and/or tables, authoredor reviewed drafts of the paper.

� Guohui Li analyzed the data, prepared figures and/or tables.� Xi Cheng performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables.

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� Muhammad Abdullah performed the experiments, analyzed the data, contributedreagents/materials/analysis tools, prepared figures and/or tables.

� Xu Sun analyzed the data, prepared figures and/or tables.� Yongping Cai conceived and designed the experiments, authored or reviewed drafts ofthe paper, approved the final draft.

� Yi Lin conceived and designed the experiments, authored or reviewed drafts of thepaper, approved the final draft.

Data AvailabilityThe following information was supplied regarding data availability:

The raw measurements are available in the Supplemental Files.

Supplemental InformationSupplemental information for this article can be found online at http://dx.doi.org/10.7717/peerj.6628#supplemental-information.

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